SUMMARY We aim to understand the nature of CD4+ T cells that protect against rhinovirus (RV) infection. Rhinovirus infection is a major cause of common cold and an important trigger of disease exacerbations among those suffering from chronic lung disorders, thereby presenting an enormous health burden to society. The knowledge gained in this project could inform the design of vaccines to prevent repeat infections and their adverse sequelae, thereby greatly impacting human health. A major challenge is to identify those CD4+ T cells that protect against the numerous different strains of RV. Our preliminary findings suggest that circulating RV- specific CD4+ T cells directed against highly conserved regions of RV capsid proteins (VP1 and VP2) protect against infection. These cells comprise a mixture of memory effector T-cell (Teff, CXCR5neg) and T follicular helper (Tfh, CXCR5+) populations that we predict will mediate viral clearance (Teff), and promote antibody production by B cells (Tfh). Here, we address the hypothesis that RV-specific Teff and Tfh cells recognizing conserved RV epitopes confer cross-protection against RV strains through maintenance of stable lineage commitment and function. In Aim 1, we will confirm that virus-specific Teff and Tfh cells induced during the effector phase of RV infection persist into the memory phase and maintain their T-cell lineage relationships. To accomplish this objective, novel MHCII tetramers displaying conserved RV-16 peptides will be used to monitor the emergence and evolution of circulating RV-16-specific T cells in healthy adults who receive experimental challenge with RV-16. High-dimensional immunophenotyping will be performed using mass cytometry coupled with advanced computational tools to visualize and track discrete RV-specific T-cell populations, as well as synchronized fluxes in B-cell populations and other cell types including CD8+ T cells, T cells, NK cells, and NKT cells. Gene expression profiling, including single-cell transcriptomics, will be integrated to test whether the degree of homogeneity within RV-specific T-cell populations is preserved into the memory phase. In Aim 2, the capacity for pre-existing RV-16-specific T-cell populations induced by primary exposure to protect against re-infection with the heterotypic strain RV-39, will be addressed using a novel sequential RV challenge model that involves re-challenge with either RV-16 or RV-39 four months after RV-16 exposure. The relationship between higher numbers of pre-existing RV-16-specific T cells and correlates of protection against RV-39, including quantitative viral shedding and production of cross-reactive serum antibodies, will be determined. Further, we will explore how homotypic and heterotypic secondary viral exposures modulate T-cell populations with protective attributes. Finally, using in vitro assays, we will confirm that Teff and Tfh cells that persist after primary and secondary RV exposures can exert cross-protective anti-viral functions. By applying a single-cell systems biology approach, the outcomes are expected to provide the groundwork for the design of T-cell vaccines for RV, and to identify new cellular and molecular targets that might be exploited for treatment.